ABSTRACT Osteoporosis is a recognized complication of inflammatory bowel disease (IBD). Aim To investigate the role of environmental factors and vitamin D receptor (VDR) variants on the prevalence of osteoporosis.
DEXA scans and case note review were performed on 440 IBD patients from 1997 to 2006. All the IBD patients and 240 healthy controls were genotyped for VDR variants Taq-1 and Apa-1 using PCR-RFLP.
Osteoporosis and osteopenia rates were 15% and 18% for IBD, 16% and 18% for Crohn's disease (CD) and 13% and 19% for ulcerative colitis, respectively. On univariate analysis of the CD patients, low body mass index (BMI, <18.5) and smoking status (P = 0.008 and 0.005 respectively) were associated with osteoporosis and osteopenia. Low BMI was also associated with osteoporosis on multivariate analysis in CD (P = 0.021, OR 5.83, CI 1.31-25.94). No difference was observed between Taq-1 and Apa-1 VDR polymorphisms in IBD, CD, ulcerative colitis and healthy controls. However, CD males were more likely to carry the variant Taq-1 polymorphism than healthy controls males (P = 0.0018, OR 1.94, CI 1.28-2.92) and female CD patients (P = 0.0061, OR 1.60, CI 1.17-2.44).
In this well-phenotyped cohort of IBD patients, a relatively low prevalence of osteoporosis was observed. Low BMI was the only independent risk factor identified to be associated with osteoporosis.

INTRODUCTIONThe inflammatory bowel diseases (IBD), Crohn’s dis-ease (CD) and ulcerative colitis (UC) are immune-mediated diseases that result in chronic, relapsinginflammation of the gastrointestinal (GI) tract. Thesediseases occur in genetically susceptible individualswho are exposed to as yet poorly defined environ-mental stimuli.1, 2Osteoporosis is defined by the World Health Orga-nisation (WHO) as: ‘A disease characterized by lowbone mass and micro architectural deterioration ofbone tissue, leading to enhanced bone fragility and aconsequent increase in fracture risk.’3The estimatedprevalence of osteoporosis in the IBD populationranges widely from 13% to greater than 50%.4–7Thiscomplication has important implications in terms ofmorbidity and population-based studies from Notting-ham, UK have shown a 60% increased risk of frac-ture in IBD patients.5The aetiology of low bone mineral density (BMD) inIBD is multifactorial. Risk factors include low bodymass index (BMI).4, 8, 9Recent data from England arepertinent. Bartram et al. in Newcastle observed that CDpatients with osteoporosis had a mean BMI of 22 com-pared with those without osteoporosis where the BMIwas 25 (P < 0.0001).4A number of studies have shownthat corticosteroid use is also a risk factor for thedevelopment of osteoporosisIBD6, 7, 10, 11and other factors that include smoking,hypogonadism, increased secretion of osteoclast stimu-lating cytokines, calcium and vitamin D deficiency,diseaselocation and durationcated.4, 7–14Twin studies have suggested that up to 80% of apatient’s BMD is genetically determined.15In view ofits chromosomal location and function, there has beensustained interest in the contribution of germline vari-ation of the vitamin D receptor (VDR) gene. The VDRgene is located within the IBD2 susceptibility locus onchromosome 12 and it spans at least 105 kb.16–18Itencodes a steroid receptor that mediates the effects of1,12(OH)2 vitamin D3 by regulating the transcriptionof cellular genes.19The gene plays a well-describedrole in skeletal metabolism but has also been shownto have immunomodulatory effects as well as possibleroles in the growth of cancer cells and in insulinsecretion.20The index study implicating VDR polymorphismsin the regulation of bone density published in Natureinpatientswithhavebeen impli-in 1994 suggested that the Bsm-1 variant of theVDR gene in intron 8 conferred 75% of the geneticvariability of BMD; however, subsequently genotyp-ing errors were identified and the results of thisstudy were modified.21Two other VDR polymor-phisms, Apa-1 and Taq-1 were found to be in stronglinkage disequilibrium with the original Bsm-1 vari-ant andtheBsm-1–Apa-1–Taq-1shown to have a moderate association with osteopo-rosis.22, 23More recently, the contribution of the VDR genewas studied in comprehensive detail by Fang et al.who observed a modest association between promotervariation and 3¢ untranslated haplotypes and fracturerisk.16These data contrast with data from a large par-ticipant level meta-analysis of European patients,where no association was observed between low BMDand any of the VDR variants examined (Cdx-2, Fok-1,Bsm-1, Apa-1, Taq-1).24In a cohort of 245 CD patients from Newcastle, UK,no association was observed between VDR variantsFok-1 and Taq-1 and low BMD25; however, in a fur-ther cohort of CD patients from Oxford, UK, there weremore VDR, Taq-1 homozygotes in the CD cohort whencompared with a healthy control population.26The aims of the present study were to investigatethe prevalence of osteoporosis in our IBD cohort, andto evaluate the contribution of specific environmentaland genetic factors on the development of osteoporosisin this IBD population.haplotypewasMETHODSPatients and controlsFour hundred and forty IBD patients attended theWestern General Hospital, Edinburgh and had demo-graphic data collected and DNA stored as part of theIBD database. All the patients selected had a confirmeddiagnosis of IBD, using the Lennard–Jones criteria.27All had undergone at least one DEXA scan. Patientsunder the age of 20 at the time of the DEXA scanwere excluded. For each patient, the following datawere generated: age, gender, age at diagnoses, diseaseduration (in years), BMI, azathioprine therapy, inflix-imab therapy, smoking status, family history, surgery,the location of disease at diagnosis and behaviour ofdisease at 5 years follow-up using Montreal classifica-tion.28Two hundred and eighty-six CD patients and154 UC patients were recruited (Table 1).LOW BMI PREDICTS OSTEOPOROSIS IN IBD589ª 2008 The Authors, Aliment Pharmacol Ther 27, 588–596Journal compilation ª 2008 Blackwell Publishing Ltd

was defined as a T-score of below )2.5 and osteopeniawhen the T-score was between )1.0 and )2.5.3Vitamin D receptor genotypingGenomic DNA was extracted from peripheral venousblood by a modified salting-out technique, and re-suspended in 1· TE [10 mM Tris (pH 8.0), 1 mM EDTA(pH 8.0)] at a final concentration of 100 ng⁄lL.29VDRTaq-1 (rs17880019) and Apa-1 (rs17879735) genotyp-ing was carried out using restriction digestion polymer-ase chain reaction (PCR). The primers used were forward5¢-CAGAGCATGGACAGGGAGCAA-3¢,TTCGAGCACAAGGGGCGTTAG-3¢. PCR conditions wereas follows – initial denaturation at 94 ?C for 4 min fol-lowed by 33 cycles of (94 ?C for 50 s, 64 ?C for 60 s,followed by 72 ?C for 90 s) and a final extension at72 ?C for 8 min and 30 s. Ten microlitres of PCR prod-uct was then added to 10 lL of Taq-1 or Apa-1 digestmixture and incubated at 65 ?C overnight. The digestionproducts were then separated on 1.5% agarose gel withethidium bromide. The gel was visualized on Genege-nius Bioimager (Syngene) with GENESNAP software usingultraviolet light. The images were recorded digitally.back5¢-CAC-Statistical analysisData were compared using the chi-squared test or whenappropriate Fisher’s exact test using MINITAB software(Minitab Ltd, Coventry, UK). The Mann–Whitney U-testwas used to compare nonparametric BMD data. To assessdifferences between steroid dosages in the osteoporoticand non-osteoporotic groups, an unpaired t-test withWelch correction was used. To identify significant inde-pendent variables associated with phenotype, multivariatelogistic regression analysis was carried out. Each singlenucleotide polymorphism (SNP) was analysed for associa-tion with IBD overall, CD, UC and disease phenotype andallele frequencies were determined for each polymor-phism. In the control group, each allele was in Hardy–Weinberg equilibrium. HAPLOVIEW software was used toestimate haplotypes (Broad Institute of MIT and Harvard,Boston, MA, USA). A P < 0.05 was considered significant.RESULTSPrevalence of osteoporosis and osteopeniaWhen all of the 440 IBD patients were investigated,15% were osteoporotic, 18% had osteopenia and 67%had normal T-scores at their lumbar spine (Figure 1).In the CD patients, 16% were osteoporotic, 18% wereosteopenic and 66% had normal T-scores at theirlumbar spine and in the UC patients, 13% were osteo-porotic, 19% were osteopenic and 68% had a normalT-score.Risk factors for reduced BMDIn CD patients, osteoporosis was associated with lowBMI (<18.5) – 44% of the low BMI patients had osteo-porosis compared with 14% of the patients with a nor-mal BMI who had osteoporosis, P = 0.048, OR 4.9 CI1.2–19.8. A linear correlation between T-score at thevertebral spine and BMI was observed in patients withCD (r2= 0.034, P = 0.0009) (Figure 2). When BMD wascompared with BMI, again a linear correlation wasobserved (P = 0.0362).Further analysis was carried out to include CDpatients at increased risk of fracture – those with oste-oporosis and osteopenia. Low BMI (P = 0.0008) andhistory of being a current or ex-smoker (P = 0.005)were associated with osteoporosis and osteopenia(Table 2). No association was observed between thenumber of months a CD patient was on corticosteroidtherapy and osteoporosis. Osteoporotic CD patients hada median of 30.4 months of corticosteroid therapy andthe non-osteoporotic CD patients had a median of29.5 months of corticosteroid therapy (P = 0.92). Nophenotypic associations were observed with osteoporo-sis in the UC cohort.0% IBD CD UC 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% OsteoporosisOsteopeniaNormalFigure 1. Low prevalence rates of osteoporosis and oste-openia were observed in patients with inflammatorybowel disease (IBD), Crohn’s disease (CD) and ulcerativecolitis (UC).LOW BMI PREDICTS OSTEOPOROSIS IN IBD591ª 2008 The Authors, Aliment Pharmacol Ther 27, 588–596Journal compilation ª 2008 Blackwell Publishing Ltd

severity and this may explain why the osteoporosislevels are lower than those reported in this series.In the same context, it is noteworthy that DEXAscans in our cohort were performed closer to the dateof diagnosis compared with previous studies. Thirty-two per cent of patients had their DEXA scan within2 years of diagnosis and the median duration to thefirst DEXA was 6 years. As the median age of diagno-sis of IBD was 29.5 years, this cohort also represents ayounger population of IBD patients compared withprevious series. Our series also includes substantiallymore patients than previous studies and this mayresult in a more accurate estimation of the prevalenceof osteoporosis.Low BMI was the strongest risk factor for osteoporo-sis in our CD population, consistent with data from anumber of previous studies.4, 6, 8, 9Despite only 3% ofpatients having a BMI of less than 18.5, this was a sig-nificant risk factor when logistic regression analysiswas undertaken. Using a multivariate model, variancein BMI accounted for approximately 8.4% of the vari-ance in BMD, a percentage lower than that suggestedby previous studies.4However, it is worth noting thatoverall only 13% of the variance in BMD wasexplained by the environmental factors we have stud-ied (including corticosteroid dose). These data wouldsuggest that, in our cohort, there are either otherunidentified critical environmental modifiers, or a crit-ically important genetic component involved in deter-mining BMD.Increasing data are emerging both in patients withIBD and in the healthy population that low BMI is asignificant independent risk factor for osteoporosis. Ina recent study of postmenopausal women in the USA,Asomaning et al. observed a significant linear trendacross BMI categories with those with the lowest BMIhaving the lowest BMD.31The authors went on toevaluate BMI as a continuous variable and observed adecrease of 12% in BMD for each point decrease inBMI. Low BMI has also been observed to predict riskof hip fracture even after adjustment for BMD. In ameta-analysis of 60 000 patients from 11 prospectivestudies, the relative risk of fracture rose from 1.4 infemales with a BMI of 20 to 2.2 in females with a BMIof 15.32In this study, being a current or ex-smoker was alsoassociated with osteoporosis and osteopenia on uni-variate analysis. The mechanisms underlying smoking-associated bone loss and fracture risk remain poorlyunderstood and previous studies have suggested thatthe effect of smoking appears to be dose-dependent,and may be reversible.33Smoking was not an indepen-dent risk factor in the multivariate analysis, however,suggesting that it plays a smaller role than low BMI indetermining BMD in the present cohort.No association was observed between corticosteroiduseand osteoporosis whenpatients were compared with a matched non-osteopo-rotic CD group. Although the role of corticosteroids inthe development of osteoporosis in patients with IBDremains controversial, this result is in line with previ-ous data published from our centre in 1994 where lowbone mineralization was observed in patients with CDat diagnosis and prior to any corticosteroid therapy.34Collating cumulative steroid exposure retrospectivelyby case notes analysis is difficult. Our design hasallowed us to address this question to a similar extentto previous studies, but we acknowledge this as apotential limitation of the present study and we feelstrongly that further prospective studies are required.In this study, we also did not collate data on the useof bisphosphonates; however, we sought to minimizethis potential confounding factor by studying a youngcohort and using the index DEXA scan for analysis.When the VDR variants Apa-1 and Taq-1 wereexamined in our cohort of IBD patients, no associationwas observed between osteoporosis and these variants.Our results are consistent with data from Newcastlewhere in patients with CD no association was observedbetween VDR variants Taq-1 and Fok-1 and osteopo-rosis.25Results from both Newcastle and our data arealso consistent with a population based Europeanmeta-analysis of 26 242 patients where no associationwas observed between any of the five VDR variantsthat were examined and low BMD.24Although Fang et al. did not observe an associationbetween VDR variants and low BMD, they did observean association with fracture risk and the authors wenton to provide functional data showing reduced VDRexpression in variant reporter assays and increasedmRNA degradation.16They further speculated that thesevariants may alter fracture risk by modifying bonemicro-architecture rather than influencing BMD. Frac-ture incidences were not measured in this study and itmay be that new imaging techniques will allow investi-gators to assess better bone micro-architecture ratherthan BMD alone to determine the fracture risk better.Interestinglyinourobserved that the Taq-1 VDR variant was moreprevalent in the male CD population compared withtheosteoporoticCDsub-group analysis,we594 C. L. NOBLE et al.ª 2008 The Authors, Aliment Pharmacol Ther 27, 588–596Journal compilation ª 2008 Blackwell Publishing Ltd

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[Show abstract][Hide abstract]ABSTRACT:
The emerging role of vitamin D as a regulator of both innate and adaptive immune responses has encouraged the investigation of its role in the pathogenesis of a variety of autoimmune conditions including the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Animal models consistently demonstrate that vitamin D significantly impacts on the modulation of astrointestinal inflammation, while epidemiological and observational data show an inverse relationship between vitamin D status and the onset/progression of Crohn's disease as well as the development of colorectal cancer. As vitamin D supplementation is readily available, at low cost, it is a very attractive potential therapeutic option. The biological plausibility for a role for vitamin D in inflammation modulation, the potential genetic links associated with vitamin D metabolism and the clinical aspects for it in IBD will be discussed.

[Show abstract][Hide abstract]ABSTRACT:
Objective
Ulcerative colitis (UC) and Crohn’s disease (CD) result from an interaction between genetic and environmental factors. Though several polymorphisms have been identified in PTPN2, their roles in the incidence of UC and CD are conflicting. This meta-analysis was aimed to clarify the impact of these polymorphisms on UC and CD risk.
Method
PubMed, EMBASE, Cochrane Library and CBM were searched until 23 July 2013 for eligible studies on three PTPN2 polymorphisms: rs2542151, rs1893217 and rs7234029. Data were extracted, and pooled odd ratios (ORs) as well as 95 % confidence intervals (95 % CIs) were calculated.
Conclusion
The meta-analysis indicated that rs2542151, rs1893217 and rs1893217 were associated with increased CD risk, while the former was associated with increased UC risk. The differences in age of onset and ethnic groups may influence the associations. Gene–gene and gene–environment interactions should be investigated in the future.
Results
Seventeen studies with 18,308 cases and 20,406 controls were included. Significant associations were found between rs2542151 polymorphism and CD susceptibility (OR = 1.22, 95 % CI, 1.15–1.30, I
2 = 32 %), as well as between rs2542151 and UC susceptibility (OR = 1.16, 95 % CI, 1.07–1.25, I
2 = 39 %). A similar result was found in Caucasians, but not in Asians. Moreover, a significant increase in CD risk for all carriers of the minor allele of rs1893217 (OR = 1.45, 95 % CI, 1.23–1.70, I
2 = 0 %) and rs7234029 (OR = 1.36, 95 % CI, 1.16–1.59, I
2 = 0 %) were found. For children, the rs1893217 polymorphism appeared to confer susceptibility to CD (OR = 1.56, 95 % CI, 1.28–1.89, I
2 = 0 %).

[Show abstract][Hide abstract]ABSTRACT:
: Technological advances in the large scale analysis of human genetics have generated profound insights into possible genetic contributions to chronic diseases including the inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis. To date, 163 distinct genetic risk loci have been associated with either Crohn's disease or ulcerative colitis, with a substantial degree of genetic overlap between these 2 conditions. Although many risk variants show a reproducible correlation with disease, individual gene associations only affect a subset of patients, and the functional contribution(s) of these risk variants to the onset of IBD is largely undetermined. Although studies in twins have demonstrated that the development of IBD is not mediated solely by genetic risk, it is nevertheless important to elucidate the functional consequences of risk variants for gene function in relevant cell types known to regulate key physiological processes that are compromised in IBD. This article will discuss IBD candidate genes that are known to be, or are suspected of being, involved in regulating the intestinal epithelial barrier and several of the physiological processes presided over by this dynamic and versatile layer of cells. This will include assembly and regulation of tight junctions, cell adhesion and polarity, mucus and glycoprotein regulation, bacterial sensing, membrane transport, epithelial differentiation, and restitution.